Producing renewable sources for a variety of fuels and chemicals is of great importance to a world with increasing demand for such products. While petroleum is a product of decayed plant and other matter that has been incubated beneath the earth's surface for millions of years, some efforts today focus on the direct use of plants and other organisms to generate, e.g., lipids, which can include fatty acids and derivatives thereof, for use in the fuel and chemical industries. Specifically, recent effort has been directed to designing algae to produce lipids for biofuel production because algae can proliferate over a wide range of environmental conditions and because algae do not compete with food crops for arable growth space. See, Hu et al. (2008) Plant J. 54, 621-39.
Algal cells are a promising source of biofuels. Wijffels & Barbosa (2010) Science 329, 796-99. Their ability to harness solar energy to convert carbon dioxide into carbon-rich lipids already exceeds the abilities of oil-producing agricultural crops, with the added advantage that algae grown for biofuel do not compete with crops for agricultural land (Wijffels & Barbosa, 2010). In order to maximize algal fuel production, new algal strains will need to be engineered for growth and carbon fixation at an industrial scale (Wijffels & Barbosa, 2010).
Triacylglycerol or triglyceride (TAG), a heterogeneous group of molecules with a glycerol backbone and three fatty acids attached by ester bonds, is an excellent molecule for high-concentration metabolic-energy storage. TAG the major form of energy storage in many eukaryotic algae under stress conditions, such as under nutrient limitation or depletion, where nitrogen depletion (where there is essentially no available nitrogen in the culture medium) is particularly effective in increasing TAG production in many eukaryotic algal species. However, culturing algae under nitrogen deficiency simultaneously limits overall lipid productivity of the culture by limiting overall biomass accumulation (Brennan and Owende (2010) Renewable and Sustainable Energy Reviews 14: 557-577). Improving the scalability, controllability, and cost-effectiveness of TAG production would be beneficial to the development of renewable energy and chemical sources.
One means of boosting TAG production is to grow algae in a two-step process alternating between nutrient-rich and nutrient-limited conditions. The nutrient-rich growth phase allows the algae to proliferate, while nutrient limitation (e.g., nitrogen depletion) results in the production of storage lipids. See, Rodolfi et al. (2009) Biotechnol. Bioeng. 102, 100-12. This process makes TAG production more expensive, because it requires long periods of growth during which the algae are producing little to no TAG.
Another means of boosting TAG production is to grow the algae heterotrophically by supplying extra organic carbon. For example, in various scenarios, organic carbon may be supplied as glycerol, one or more sugars, one or more organic acids, or other reduced carbon sources added to the growth medium. See, Allnutt et al. (WO 2011/026008). This heterotrophic growth technique not only increases the expense of TAG production, it also risks the contamination of the algal cultures with exogenous bacteria or fungi whose growth can be stimulated by the added carbohydrates. See, Scott et al. (2010) Curr. Opin. Biotechnol. 21, 277-86.
The biosynthesis pathways leading to the production of TAG have been studied. In the final reaction of the Kennedy pathway, diacylglycerol (DAG), a precursor to both membrane and storage lipids, is covalently linked to a fatty acyl to produce TAG. This reaction is catalyzed by the diacylglycerol acyltransferase (DGAT) enzyme (Kennedy (1961) Fed. Pro. Fed. Am. Soc. Exp. Biol. 20, 934-40). There are two distinct gene families in eukaryotic organisms which encode enzymes which catalyze this reaction, DGAT1 and DGAT2, which have little sequence similarity. Evidence from higher plants as well as mammals suggests that the two gene families have different functions, although the exact role of each type of DGAT has not been elucidated, and may differ in different species (Yen et al. (2008) J. Lipid Res. 49, 2283-301). A third DGAT gene family known as DGAT3 genes encode soluble DGATs, such as that of peanut (Saha et al. (2006) Plant Physiol 141: 1533-1543) and Arabidopsis (Hernandez et al. (2012) Plant Physiol. Published on Jul. 3, 2012, as DOI:10.1104/pp. 112.201541). Another member of the extended DGAT family is the diacylglycerol acetyl-CoA transferase that transfers a two carbon acetyl group, rather than a longer acyl chain, to DAG (Durrett et al. (2010) Proc. National Acad Sci USA 107: 9464-9469). Additionally, certain prokaryotic species that are able to accumulate neutral lipids include acyltransferases for the production of wax esters or TAG that belong to the “WS/DGAT” family of DGATs (Barney et al. (2012) Appl. And Environ Microbiol. 78: 5734-5745).
Although overexpression of DGAT genes was found in several studies to increase TAG accumulation in higher plants, as yet attempts to increase TAG production by overexpression of DGAT genes in eukaryotic algae have been unsuccessful (Courchesne et al. (2009)).
Roberts et al. (U.S. Pub. No. 2010/0255551) and Roberts et al. (U.S. Pub. No. 2010/0184169) report the expression of DGATs derived from Acinetobacter baylii, Streptomyces cœlicolor, and Alcanivorax borkumensis in the cyanobacteria Synechococcus elongatus and Synechocystis PCC 6803.
Benning et al. (U.S. Pub. No. 2010/0192258) disclose DGAT genes from the alga Chlamydomonas reinhardtii and report their expression in Saccharomyces cerevisiæ. 